Mammalian Cell Culture
The process of mammalian cell culture has been refined and standardised over many years and the conditions required to permit cell growth, expansion and differentiation are well established. Cells can be routinely cultured on sterile plastic surfaces, such as polystyrene, often in conjunction with a matrix coating designed to more closely replicate the in vivo environment in which the cell would normally grow. In the case of embryonic stem cells, a layer of feeder cells such as mouse embryonic fibroblasts is often required to provide a better substrate to nourish and support the proliferation of the cells. Typically, mammalian cells will be cultured in a supportive medium usually containing additives such as foetal calf serum, which in addition to providing various hormones and growth factors to the cells also contains a number of cellular matrix components which again helps to replicate the ideal microenvironment for cell growth.
Currently, mammalian cell culture has a variety of downstream applications including their use in basic research, their use in drug and toxicology assays and their use in producing correctly folded recombinant mammalian proteins. More recently mammalian cell culture has been further developed to be used in the area of regenerative medicine, whereby tissue, such as skin dermal layers, is harvested and propagated for a certain time period in order to generate larger amounts of tissue for use in clinical surgery, e.g. repair of wounds after traumatic injury or repair of diseased internal organs. However, cells derived from primary sources are often very limited in their capacity to be further propagated and require complex formulations of growth medium and growth conditions to be successfully cultured. They also have limited use in regenerative medicine due to the problem of immune rejection.
Recently, there have been a number of developments in the field of embryonic stem cell research which aim to traverse these barriers. Not only can such pluripotent stem cells be propagated almost indefinitely, they also have the capacity to differentiate into every cell and tissue type which comprise the fully developed organism. As such, pluripotent stem cells can be an important tool in developing new drug assays and since their supply is virtually unlimited they provide a cheaper and less variable source.
3D Bioscaffolds
One of the main challenges in differentiating pluripotent stem cells has been to control differentiation in such a way as to consistently produce mature, fully functional cell types rather than partly differentiated, immature precursor cells or highly heterogeneous mixtures of various cell types. The simplest way to differentiate pluripotent stem cells is on a 2D plastic substrate but unfortunately this often produces differentiated cells which lack the characteristic phenotype of mature cell types. This is in part due to a failure to replicate the conditions experienced by stem cells during normal embryogenesis, a process which differs from 2D culture in that it does of course take place in 3 dimensions. One of the ways researchers have attempted to address this has been to develop novel 3D culture systems which aim to more faithfully replicate some of the conditions experienced by cells during embryogenesis and allow them to differentiate in a more natural way. In particular, such 3D systems allow cells to interact with each other to a greater degree and allow the development of complex multicellular aggregates more akin to functioning organs than anything that can be observed using 2D cell culture.
3D systems rely on the presence of a bioactive scaffold onto which the cells can be seeded and to which they can adhere. Such a scaffold must help to direct the growth and proliferation of cells in a desired 3D configuration and may also be required to provide certain molecular signals which help the cells to form the desired structures. Another important requirement of bioscaffolds is that they are scalable, so that tissue growth and cell differentiation can be carried out on a larger, more economical scale. Scaffolds may be composed of a variety of materials and correct scaffold selection may be crucial in directing the growth, proliferation and differentiation of any cells which are seeded onto it. For example, scaffolds are commonly composed of polymeric materials which are arranged into the form of a porous sponge. Cells seeded into this scaffold can then attach and grow inside the pore structure of the scaffold through the network of interconnecting tunnels and channels inside the scaffold, with pore size being an important consideration when selecting an appropriate scaffold. Bioactive agents, such as extracellular matrix (ECM) components, may also be used to enhance scaffold function when deposited onto a scaffold surface, permitting greater cell adhesion. The end result is to provide cells with an in vitro environment in which they can interact more realistically and in a manner which more closely resembles their normal in vivo home.
In several culture systems, the addition of extracellular matrix induces cellular polarity and tissue organisation. For example, when a monolayer of primary hepatocytes cultured on a flat sheet of collagen is further overlaid with a second layer of collagen, a so called sandwich culture, the cells show improved morphology and functionality compared to hepatocytes in conventional 2D cultures (Dunn, J. et al., 1991). The overlay causes the cells to maintain actin filaments similar to the in vivo state in contrast to the abnormal formation of stress fibres seen in the 2D control culture (Berthiaume, F. et al., 1996). The more physiological relevant cytoskeletal organisation and the following cell polarity and shape might be responsible for the improved hepatic functionality, although it was later argued that the beneficial effects of the sandwich culture primarily arise from the improved cell-cell contact rather than the cell contact to the extracellular matrix (Hamilton, G. et al., 2001). Collagen scaffolds has also been employed in the differentiation of embryonic stem cells into hepatocytes. Baharvand and co-workers found that differentiation of hepatocyte-like cells inside collagen scaffolds improved morphological features, gene expression pattern and metabolic activity compared to cells differentiated in traditional 2D (Baharvand, H. et al., 2006).
Alginate, which provides a porous non-adhesive 3D-scaffold support aggregation of hepatocytes to form strong cell-cell interaction leading to improved hepatic function (Dvir-Ginzberg, M. et al., 2003). The environment in the alginate matrix also supported differentiation of newborn rat hepatocytes (Dvir-Ginzberg, M. et al., 2008) and maturation of HepG2 (Elkayam, T. et al., 2006) into more mature, functional phenotypes. Additionally, the C3A hepatic cell line showed improved drug metabolism as enzymatic activity of a number of different CYPs where increased when cultured as spheroids in alginate compared to in 2D monolayer cultures (Elkayam, T. et al., 2006). Recently several new products for providing 3D scaffolds to support cell culture have been launched on the market, including 3D interweaving nanofibre scaffolds which have been found to improve hepatic culture conditions (Wang, S. et al., 2008). Alternatively, porous polystyrene scaffolds provides space to enable cells to grow and differentiate and form layers that develop complex 3D cell-cell interactions (Bokhari, M. et al., 2007a; Bokhari, M et al., 2007b). In these scaffolds, HepG2 respond to biochemical agents in a manner much more resembling the activity of tissues in vivo compared to cultured in 2D (Bokhari, M. et al., 2007a). Additionally, mouse ES cells have successfully been differentiated as spheroids in perfused polyurethane foam towards hepatocytes, aiming to develop a mass differentiation culture method by combining growth factor treatment with multicellular spheroid formation (Matsumoto, K. et al., 2008).
Hepatocyte Cell Culture
Liver failure and end-stage liver diseases are responsible for a huge amount of deaths around the world and is a major burden on the health care system. Liver transplantation remains the most successful treatment. However, the efficacy of this procedure is limited and connected to many complications such as infection or rejection. Liver transplantation also suffers from shortage of available donor organs and the treated patients will very often be referred to lifelong immunosuppression therapy. By reducing the need for organs, cell-based treatment will be of great importance to both society and to the individuals suffering from these severe diseases.
Furthermore, the liver is the centre of metabolism and detoxification in the human body, and therefore huge efforts have been undertaken in order to identify a reliable source of functional cell types for in vitro testing. Unfortunately, the complexity and function of the liver is not mirrored by any cell type available today.
Methods for generation of hepatocyte-like cells from hPS cells, which may be further differentiated into mature hepatocytes, often includes the formation of embryoid bodies and/or early selection based on addition of cytotoxic compounds (Rambhatla, L. et al., 2003). These selection steps, especially formation of embryoid bodies, often results in a major cell number loss and in turn low efficiency. The methods are complicated, most having very long generation times and involve several time consuming steps. Thus, there is a need for rapid and simple method for the formation of hepatocyte-like cells derived from undifferentiated hBS cells. Previous attempts to obtain hepatocyte-like cells as e.g. published in US 20030003573 results in a low yield in relation to the starting material. Furthermore, the availability of primary human liver cells is very limited and the cells are also known to rapidly loose their normal phenotype and functional properties when used for in vitro applications. One often used alternative to primary cells are hepatic cell lines which in turn contain very low levels of (or totally lack) metabolising enzymes and have distributions of other important proteins substantially different from the native hepatocyte in vivo. Thus, many tests are still performed using animal material, even though liver metabolism is known to be species specific and thereby generating difficulties in predicting liver metabolism and toxicity in other species than the one tested.
In pharmaceutical development, adverse liver reactions remain the most prominent side effect. Therefore early prediction of human liver toxicity liabilities is of paramount importance when selecting compounds to enter clinical trials. Efforts to improve capabilities in this area must address both the availability question and development of models, which provide greater coverage for the complex biological processes which coincide to induce adverse liver injury in humans.
Accordingly there is an urgent need for a model system that mimics human liver cells and that is able to predict effects of candidate molecules in the development of new drugs or chemicals. Regarding both availability and physiological relevance, hPS cells may serve as an ideal renewable source of functional human hepatocytes.